Abstract: Objective To isolate and steadily culture kidney stem cells (KSCs) from rat renal papilla, and to identify the biological characteristics of KSCs. Methods KSCs were isolated from the tips of renal papilla in 4 weeks-old Sprague-Dawley rats. The morphology of KSCs was observed under inversion microscope, and the phenotye characteristics of KSCs were identified through flow cytometry and immunofluorescence. The abilities of KSCs in adipogenic and osteogenic differentiation were evaluated. The differences of gene expression between KSCs and rat renal tubular epithelial cells （RTECs）were compared using quantitative real time polymerase chain reaction(qRT-PCR). Results KSCs showed a spindle-shaped and arborization-like growth pattern. Immunofluorescence indicated that KSCs staining with alpha-sooth muscle actin (α-SMA), Vimentin, N-Cadherin, Nestin, CD133 marker, and without E-cadherin, cytokeratin-18 (CK-18), zona occludens protein-1 (ZO-1).The positive staining of CD29, CD90, CD73 were 99.0%,95.8%,99.9% respectively,the positive staining of CD45 was 3.4%. The positive stainings of stem cell marker CD133 and Nestin were 33.2% and 70.2% respectively, while the double staining rate was 31.4%., KSCs showed positive staining by oil red O after adipogenic differentiation, and orange calcium deposition by alizarin red staining after osteogenic differentiation. qRT-PCR showed that the expressions of embryonic stem cell marker Nanog, Oct4/pou5f1,Sox2/sry-box-2 in KSCs were higher than those in RTECs （ P＜0.01）,and the expressions of mesenchymal marker α-SMA, Vimentin were also higher in KSCs （ P＜0.01）. Compared with RTECs, the expressions of mature epithelium marker E-Cadherin, CK18 in KSCs were lower （ P＜0.01）. Conclusion KSCs were isolated successfully and steadily cultured from the rat renal papilla, which were identified with featured biological characteristics.